The Molecular Modeling Core collaborates with experimental groups by providing a physical molecular, structural basis for evaluating and understanding their results. This in turn provides the grist for developing new hypotheses and experiments. The Core enjoys a long-standing collaboration with Dr. Ettore Appellas section in the Lab of Cell Biology, which has focused on understanding the molecular interactions that regulate the p53 protein. Human p53 is a homotetrameric, sequence-specific transcription factor that has crucial roles in apoptosis, cell cycle arrest, cellular senescence, and DNA repair. It is maintained at low levels in unstressed cells, but is stabilized and activated following DNA damage through extensive post-translational modification. One mechanism of initiating p53 activity is by site-specific recruitment of the p300 histone acetyltransferase coactivators, which promote local chromatin unwinding. To understand this recruitment, we have recently determined the structure of the complex formed between the N-terminal transactivation domain of p53 and the Taz2 domain of p300, and studied how site-specific phosphorylations of p53 lead to stronger binding. Furthermore, we identified a second binding site for Taz2 within p53 residues 35-59. This second site bound Taz2 with a similar affinity as the first site, but the binding was unaffected by phosphorylation. This year we have focused on determining the structure of this second transactivation domain of p53 bound to Taz2, which is nearly completed. Comparison with the structure of the first transactivation domain will further elucidate how the activity of p53 is regulated by Taz2. We have also been focusing of the activity of the Wip1 phosphatase. Experiments from the Appella group suggest that over-expression of Wip1 promotes tumorigenesis through inactivation of p53. Wip1 is a conserved PP2C phosphatase expressed at low levels in most tissues and transcriptionally induced after DNA damage in a p53-dependent manner. Wip1 substrates discovered thus far are p53, p38MAPK, UNG2, Chk1, Chk2 and ATM. In previous years we revealed the molecular bases for the ability of Wip1 to dephosphorylate serine and threonine residues within pTXpY and pT/SQ amino acid sequence motifs. Based on this, we have recently designed, synthesized and tested several peptide, and now, small molecule inhibitors specific for Wip1. The latter is composed of a pyrrole-based scaffold, from which extend functional groups that mimic the three-dimensional arrangement of polar and hydrophobic residues of the native peptide substrates. This year we have focused on optimizing our cyclic peptide inhibitor, which has resulted in a 30-fold increase in activity. We are currently pursuing X-ray crystallographic determination of the Wip1/inhibitor complex structure to facilitate further optimization of the binding selectivity and affinity. We hypothesize that treatment of cancers that over-express Wip1 with a Wip1 inhibitor will lead to increased activation of p53 and subsequent cell killing. Such an inhibitor would provide selective targeting of tumors either when given alone or in combination with standard cancer chemo- or radio-therapy. In a similar vein, we have also recently developed a peptide-mimetic inhibitor of both the human MDM2 and MDMX proteins (HDM2 and HDMX). Both these proteins bind to the N-terminal, transactivation domain of p53, and cause its degredation. Following our earlier work developing a peptoid (poly N-substituted glycine) inhibitor of MDM2, we have succeeded in producing a smaller and easier to synthesize inhibitor based on a polyamine backbone. This molecule has a binding affinity in the range of the well-known HDM2 inhibitor Nutlin being developed by Hoffman-La Roche. However, unlike Nutlin, our lead molecule is potent against both HDM2 and HDMX, two important targets for cancer therapy. This year we incorporated the lessons learned into a new, relatively easy to synthesize, cyclic-di-peptide inhibitor with enhance activity.